Herpes Simplex Virus 1 (HSV-1) is a significant human pathogen, and is the leading cause of blindness in the US due to an infectious agent. HSV-1 establishes a lifelong latent infection in host sensory neurons, where lytic gene expression is repressed. Latent HSV-1 periodically reactivates, and recurrences are often associated with HSV-1-related disease. It is now widely recognized that the LAT promoter and enhancer and the immediate early (IE) genes of HSV-1 are regulated through the deposition and maintenance of chromatin. However, the mechanisms involved in directing chromatin deposition during latency have not been defined, and the identification of the mechanism regulating the latent-lytic switch during reactivation remains elusive. A major focus of our research is to overcome these significant gaps in our knowledge so that novel HSV-1 therapeutic interventions can be developed in the future. Recently, we identified seven binding motifs for the cellular insulating protein CTCF. The genomic locations of each motif in HSV-1 show that CTCF binding domains flank the LAT and each IE region of the genome separately, providing evidence that CTCF insulators could control latent chromatin recruitment and HSV-1 gene expression. We showed that site was occupied by the insulator protein CTCF during latency, three sites function as enhancer-blocking insulators, and that CTCF was evicted at early times in reactivation. We subsequently pioneered a gene delivery method to show that CTCF depletion drives HSV-1 reactivation in vivo. CTCF insulators regulate gene expression through multiple mechanisms, one of which is the formation of higher order chromatin structures known as chromatin loops. Chromatin loops bring enhancers and promotors into close spatial proximity to regulate gene expression. Using deep sequencing on latently infected mouse neurons (3C-seq) we found 3 distinct chromatin loops. We are the first investigators to discover chromatin loops in an alpha-herpes virus. Consequently, each of these loops adopt a 3D conformation that orient the IE genes as ?off? or ?poised for reactivation?. We hypothesize that the three loops in HSV-1 1) control the chromatin architecture by recruitment of co-regulating proteins, and; 2) populate neuronal subtypes that can (or cannot) reactivate, based on the loop conformation. The aims of this project methodically test how the 3D orientation of these CTCF loops in HSV-1 control lytic, latent and reactivating genomes. We have pioneered novel techniques to test this hypothesis using in vivo models of HSV-1 infection, making our study not only relevant on a molecular level, but physiologically relevant as well, and our expertise in epigenetic regulation of DNA viruses make us the ideal investigators to carry out these studies. In summary, we will push the field of epigenetic control of herpesviruses into areas that have never been explored by combining genetics, molecular biology and in vivo models together to achieve comprehensive, methodical and meticulous evaluations of how the viral transcriptional program is controlled by CTCF-chromatin loops at all stages of the virus life cycle.